There are growing demands for 3D structure determination of nucleic acids and protein-nucleic acid complexes for understanding disease mechanisms at the molecular level, thus facilitating new drug discoveries. X-ray crystallography is one of the most powerful tools for structure determination of these macromolecules and complexes. However, crystallization and phase determination have been the bottleneck problems that largely slow down structural determination of new structures and folds of RNAs and protein-nucleic acid complexes. Though the approach of the nucleic acid bromination is routinely used for phasing, the bromo-derivatives often suffer from the stability issue, perturbation, crystallizability, and derivatization site limitation. Therefore, the novel technologies that facilitate crystallization and phasing are of tremendous value. The selenium replacement of sulfur in proteins has helped to revolutionize protein crystallography via selenium MAD phasing. Recently the applicant has pioneered the selenium replacement of oxygen in nucleic acids for structure and function studies. Their research is based on their central hypothesis: selenium can be used to stably replace oxygen of nucleic acids atom-specifically without significant perturbation, because selenium and oxygen are in the same elemental family. They have successfully demonstrated that the selenium derivatization of nucleic acids can solve the phase problem. Excitingly, they have also found that the Se-derivatization can facilitate crystallization of RNAs, DNAs, and protein-nucleic acid complexes. Thus, this proposed project seeks to innovatively shift the current paradigms on protein/nucleic acid crystallography by incorporating the selenium derivatization into nucleic acids and protein-nucleic acid complexes in order to routinely solve crystallization and phasing problems. The applicant has also demonstrated that the multiple Se-derivatizations do not cause significant structural perturbation in nucleic acids and protein-nucleic acid complexes. Thus, the applicant proposes to synthesize the novel phosphoramidites and triphosphates derivatized with the multi-Se-modifications (Se-clusters) for chemical and enzymatic synthesis of Se-DNAs and Se-RNAs. The multi-Se-modifications can serve as the powerful Se-derivatizing clusters for the crystallization and phasing. They will also investigate these synthesized Se-nucleic acids biophysically and structurally for crystallization, phasing, and structure determination. Furthermore, the applicant plans to study the mechanisms of crystallization facilitation. Their novel Se-derivatization technology is extremely valuable to high-throughput structural determination of nucleic acids (such as non-coding RNAs) and protein-nucleic acid complexes. Their long-term goal is to establish the novel technologies that will revolutionize crystallization, phasing, and structure determination of nucleic acids and protein-nucleic acid complexes. PUBLIC HEALTH RELEVANCE: Selenium-derivatized nucleic acids (SeNA) have great potentials in crystallization facilitation, rational phasing, and high-throughput crystal structure determination of nucleic acids and protein-nucleic acid complexes, which provides insights into molecular-level disease mechanisms and leads to new drug discoveries and disease treatments.